BR112014023414B1 - RECOMBINANT EQUINE HERPESVIRUS-1 VACCINE CONTAINING A MUTANT C GLYCOPROTEIN AND ITS USES - Google Patents

Abstract

RECOMBINANT EQUINE HERPESVIRUS-1 VACCINE CONTAINING A MUTANT C-GLYCOPROTEIN AND ITS USES. The present invention provides compositions or vaccines that contain a recombinant EHV-1 that elicit an immune response in animals against equine herpesvirus, including compositions comprising said recombinant EHV-1, methods of vaccination against equine herpesvirus, and kits for use in such methods and compositions.

Description

CROSS REFERENCE TO RELATED ORDERS

[0001] This application claims the benefit of US Provisional Patent Application No. US 61/613,151, filed March 20, 2012.

FIELD OF THE INVENTION

[0002] The present invention relates to compositions or vaccines for combating equine herpesvirus infections in animals. Specifically, the present invention provides compositions or vaccines that contain a recombinant EHV-1 that elicit an immune response in animals against equine herpesvirus, including compositions comprising said recombinant EHV-1, methods of vaccination against equine recombinant herpesvirus, and kits. for use in such methods and compositions.

BACKGROUND OF THE INVENTION

[0003] Equine herpesvirus type 1 (EHV-1) is one of the most important and prevalent pathogens of equine populations worldwide (Ma et al, J. of General Virology 91, 1817-1822, 2010). Together with its closest varicella-zoster virus, bovine herpesvirus type 1, pseudorabies virus, and EHV-4, EHV-1 form the genus Varicellovirus in the subfamily Alphaherpesvirinae of the family Herpesviridae of the order Herpesvirales (Davison et al., The order Herpesvirales. Arch Virol 154, 171-177, 2009). Diseases caused by EHV-1 range from mild rhinopneumonitis and abortion in pregnant mares to neurological disease that is often lethal in horses (Allen et al, Prog Vet Microbiol Immunol 2, 78-144, 1986; Carroll et al, Aust Vet J 62, 345-346, 1985; Crabb et al, Adv Virus Res 45, 153-190, 1995). The pathogenesis of EHV infection is very complex. Natural infection occurs through inhalation or ingestion of the infectious virus. Within a few days, the virus can be found on leukocytes, where it is protected from recognition and attack by the immune system. The virus disseminates through cell-associated viremia to secondary replication sites (Allen et al, Proceedings 8th Equine Infectious Disease Conference, Dubai 23-26, pp 129-146, 1998).

[0004] EHV-1 harbors a 150 kb double-stranded DNA genome that is highly conserved across strains. The neuropathogenic Ab4 strain (GenBank # AY665713) and a non-neuropathogenic V592 strain (GenBank accession number AY464052) have been extensively characterized and shown a nucleotide variation rate of about 0.1% (Nugent et al., J. Virol 80, 4047-4060, 2006). Only a minority of EHV-1 strains have been found to be capable of inducing neurological disorders, although all strains can cause abortion and respiratory disease (Mumford et al, J. Reprod Fertil Suppl 35, 509-518, 1987; Ostlund , Vet Clin North Am Equine Pract 9, 283-294, 1993; Wilson, Vet Clin North Am Equine Pract 13, 53-72, 1997). Recently, epidemiological studies as well as reverse-genetics studies have shown that a single -nucleotide polymorphism at position 2254 (G/A2254) of open reading frame 30 (ORF30), encoding viral DNA polymerase (Pol), will lead to a variation in the position of the amino acid 752 (D/N752), which is associated with virus neuropathogenesis (Goodman et al, J Biol Chem 281, 18193-18200, 2007; Van de Walle et al, J. Infect Dis 200, 20-25, 2009; Smith et al, Vet.Microbiol., 141, 5-11, 2010 ). It has been shown that residue 752, essentially Pol is not required of EHV-1 for virus growth and that the N752 mutation confers a drug-sensitive phenotype to the virus (Ma et al, 2010).

[0005] EHV-1 glycoprotein C (gC) has been shown to play an important role in the early steps of infection and in the release of viral particles (Osterrieder, Virus Research 2, 165, 1999). EHV-1 glycoprotein C is not essential for virus growth. It mediates primary binding, and is necessary for viral replication in equine primary cells (Osterrieder, 1999).

[0006] Conventional inactivated vaccines offer only partial clinical and virological protection against respiratory infections, but do not prevent cell-associated viremia. Considering the susceptibility of animals, including humans, to herpesvirus, a means of preventing herpes virus infection and protecting animals is essential. Therefore, there is a need for an effective vaccine against the herpes virus.

[0007] Citation or identification of any document in this patent application is not an admission that such document is available as prior art to the present invention.

SUMMARY OF THE INVENTION

[0008] The invention provides a composition or vaccine that contains a recombinant Equine Herpesvirus-1 (EHV-1). In particular, the present invention provides a recombinant EHV-1 that contains a mutant glycoprotein C (gC) gene that is non-functional. The EHV-1 recombinant gC gene can be excluded. The gC gene may encode a mutated gC protein, in which the N-terminal region of the gC protein is deleted. The recombinant EHV-1 may further comprise a DNA polymerase (Pol) gene encoding a Pol having an asparagine (N) at amino acid position 752. The recombinant EHV-1 may comprise a DNA polymerase (Pol) gene. which encodes a Pol having an asparagine (N) at amino acid position 752. The EHV-1 may be an EHV-1 RACL strain.

[0009] The invention provides methods for inducing an immunogenic or protective response against EHV-1, as well as methods for preventing EHV-1 or disease state(s) caused by EHV-1, comprising administering the composition or vaccine of the present invention. The invention also provides methods of vaccinating an animal, which comprises at least one administration of the composition or the recombinant EHV-1.

[0010] These and other embodiments are disclosed or are obvious and encompassed by the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The following detailed description, given by way of example, and which is not intended to limit the invention to the specific embodiments described, may be understood in conjunction with the accompanying figures, incorporated herein by reference, in which:

[0012] Figure 1 represents the table showing the corresponding SEQ ID NO assigned to polynucleotide and protein sequences.

[0013] Figures 2A-2C illustrate the cloning scheme. Figure 2D shows the amino acid mutation in EHV-1 polymerase.

[0014] Figures 3A-3B show the indirect immunofluorescence (IF A) reaction of RK13-Pol and RK13.

[0015] Figures 4A-4B show the growth of LI l-EYFP in R 13-Pol v. LI l-EYFP in RK13.

[0016] Figures 5A-5F depict IF A of RacL1 1, L1 and L1 l_D752NAgC 1_D752NAgC rev using anti-EHV-1 gC MAb.

[0017] Figure 6 shows the in vitro growth of RacL11, L11 D752N, L1 and 1_D752NAgC L1 1_D752NAgC rev measured by attached assay.

[0018] Figure 7 shows the in vitro growth of RacL1 1, L1 1_D752N, L1 and 1_D752NAgC L1 1_D752NAgC rev determined by comparing plate sizes.

[0019] Figure 8 shows the growth kinetics of RacL1, L11 D752N, L1 and 1_D752NAgC L1 1_D752NAgC rev by extracellular and intracellular titers.

[0020] Figures 9A and 9B show the significant virus titers in lungs infected with RACL 11, LI 1_D752N, LI 1_D752NAgC, LI 1_D752N AgC REV 2 and PBS (control) 2 and 4 days post infection (pi), 2 and 4 days after challenge (pc).

[0021] Figure 10 illustrates the histopathological changes of the lungs of mice infected with RacLl its 1_D752N read, read l_D752NAgC, LI 1_D752N AGC rev on day 2 plus

[0022] Figures 11A-11B depict serology against EHV-1 using complement fixation (CF) and virus neutralization (VN) tests.

[0023] Figure 12 shows the duration of viremia in vaccinated horses.

[0024] Figure 13 shows viral shedding in nasal swab of vaccinated horses.

[0025] Figures 14A-14C show the polynucleotide and protein sequences of EHV-1 DNA polymerase alignments.

[0026] Figures 15A-15B show the alignments of polynucleotide sequences and protein sequences of EHV-1 glycoprotein C.

[0027] Figure 16 shows the DNA and protein sequences.

DETAILED DESCRIPTION OF THE INVENTION

[0028] Note that in this specification and particularly in the claims, terms such as "comprises", "comprises", "comprising" and the like may have the meaning ascribed to them in United States patent law ; for example, they can mean "includes", "includes", "including", and the like; and that terms such as "consisting essentially of" and "consisting essentially of" have the meaning given to them in United States Patent law, for example, they allow for elements not explicitly cited, but exclude elements that are found in the prior art or that affect a basis or novel feature of the invention.

[0029] Unless otherwise indicated, technical terms are used in accordance with conventional usage. Definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes V. published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (Eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: A Global Tourism Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

[0030] The singular terms "a", "an", and "the" include plural referents unless the context clearly indicates otherwise. Likewise, the word "or" is intended to include "and" unless the context clearly indicates otherwise. The word "or" any member of a special list and also includes any combination of members of that list.

[0031] The term "N strain of EHV-1" as used herein refers to any strain of EHV-1, which has an asparagine (N) at amino acid position 752 of its DNA polymerase. The EHV-1 strain cannot be a wild-type strain comprising a DNA polymerase comprising an asparagine (N) at amino acid position 752 N The strain may be mutated or recombinant 1-EHV from EHV-1 strain, wherein the polymerase DNA is engineered to have an asparagine (N) at amino acid position 752.

[0032] The term "Kappa D EHV-1" as used herein refers to any strain of EHV-1 which has an Aspartic Acid (D) at amino acid position 752 of its DNA polymerase. EHV-1 strain D may be a wild-type strain comprising a DNA polymerase comprising an aspartic acid (D) at amino acid position 752 D. The EHV-1 strain may be a mutant or recombinant of EHV-1, wherein the DNA polymerase strain was designed to have an aspartic acid (D) at amino acid position 752.

[0033] By "animal" is meant mammals, humans, birds, and the like. The animal can be selected from the group consisting of equine (e.g. horse, zebra, donkey), canine (e.g. dogs, wolves, foxes, coyotes, jackals) feline (e.g. lions, tigers, domestic cats , wild cats, other big cats and other felines including leopards and lynx), sheep (e.g. sheep), cattle (e.g. cattle, cow, buffalo), swine (pig), poultry (e.g. chicken, duck, goose, turkey, quail, pheasant, parrot, finches, hawk, crow, ostrich, emu, and cassowary), primate (eg, prosimian, tarsier, monkey, gibbon, monkey), and fish. The term "animal" also includes an individual animal at all stages of development, including embryonic and fetal stages.

[0034] The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of consecutive amino acid residues.

[0035] The term "nucleic acid", "nucleotide" and "polynucleotide" refers to RNA or DNA and its derivatives, such as those containing modified backbones. It should be appreciated that the invention provides polynucleotides comprising sequences complementary to those described herein. The polynucleotides according to the invention can be prepared in different ways (for example, by chemical synthesis, by gene cloning, etc.) and can take different forms (for example, linear or branched, single or double-stranded, or a your hybrid, primers, probes, etc).

[0036] The term "gene" is used broadly to refer to any polynucleotide segment associated with a biological function. Thus, genes or polynucleotides include introns and exons as in the genomic sequence, or just the coding sequences, as in cDNA, such as an open reading frame (ORF), starting from the initiation codon (methionine codon) and ending with a termination signal (stop codon). Genes and polynucleotides may also include regions that regulate their expression, such as transcriptional initiation, translation, and transcriptional termination. Thus, promoters and ribosome binding regions are also included (in general these regulatory elements are found approximately between 60 and 250 nucleotides upstream of the initiation codon of the coding sequence or gene; Doree SM et al.; Pandher K et al. .; Chung et JY al), transcriptional terminators (generally the terminator is located within approximately 50 nucleotides downstream of the stop codon of the coding sequence or gene; CK Ward et al.). Gene or polynucleotide also refers to a nucleic acid fragment that expresses functional mRNA or RA, or encodes a specific protein, and that includes regulatory sequences.

[0037] As used herein, the term "antigen" or "immunogen" designates a substance that induces a specific immune response of a host animal. The antigen may comprise a whole organism, dead, attenuated or alive; a subunit or part of an organism; a recombinant vector containing an insert that expresses an epitope, polypeptide, peptide, protein, or a fragment thereof with immunogenic properties; a nucleic acid part or fragment capable of inducing an immune response against presentation to a host animal; a protein, a polypeptide, a peptide, an epitope, a hapten, or any combination thereof. Alternatively, the immunogen or antigen may comprise a toxin or antitoxin.

[0038] By definition, an epitope is an antigenic determinant that is immunologically active in the sense that, once administered to the host, it is capable of evoking a humoral (B-cell) and/or cell-type (T-cell) immune response. ). These are chemical groups or peptide sequences on a molecule that is antigenic. An antibody specifically binds to a specific antigenic epitope of a polypeptide. As specific, non-limiting examples of an epitope include tetra to penta peptide sequence in a polypeptide, a tri- to penta-glycoside sequence in a polysaccharide. In most animals, antigens will present several or even many antigenic determinants simultaneously. Such a polypeptide can also be classified as an immunogenic polypeptide and the epitope can be identified as described below.

[0039] An "isolated" biological component (such as a nucleic acid or protein or organelle) refers to a component that has been substantially separated or purified from other biological components in the cell of the organism in which the component naturally occurs, e.g. example, other chromosomal and extrachromosomal DNA and RNA, proteins, and organelles. Nucleic acids and proteins which have been "isolated" include nucleic acids and proteins purified by conventional purification methods. The term also encompasses nucleic acids and proteins prepared by recombinant technology, as well as chemical synthesis.

[0040] The term "purified" as used herein does not require absolute purity; on the contrary, it is conceived as a relative term. Thus, for example, a purified polypeptide preparation is one in which the polypeptide is more enriched than the polypeptide that is in its natural environment. A polypeptide preparation is substantially purified such that the polypeptide represents several embodiments, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98%, of the total polypeptide content of the preparation. The same applies for polynucleotides. The polypeptides described herein may be purified by any of the means known in the art.

[0041] The present invention provides a composition or a vaccine comprising an equine Herpesvirus-1 (EHV-1) recombinant viral vector. In one aspect, the present invention provides a recombinant EHV-1 that comprises a mutant glycoprotein C (gC) gene. In another aspect, the present invention provides a recombinant EHV-1 which comprises a DNA polymerase having an asparagine (N) at amino acid position 752. In yet another aspect, the present invention provides a recombinant EHV-1, wherein the EHV -1 is an N-strain EHV-1. The recombinant EHV-1 N-strain may comprise a mutant gC gene. In yet another aspect, the recombinant EHV-1 comprises a mutated gC gene and a DNA polymerase that has an asparagine (N) at amino acid position 752. The composition or vaccine of the present invention may further comprise a carrier, diluent, adjuvant or pharmaceutical or veterinarily acceptable excipient.

[0042] The term "composition" includes any vaccine or immunological composition, once injected into a host, including canine, feline, equine and human, that induces an immune response in the host, and/or protects the host against leukemia and/or which may prevent parasite implantation and/or which may prevent disease progression in infected individuals and/or which may limit the spread of fugitive parasites from internal organs. This can be accomplished after vaccination in accordance with the present invention, by inducing cytokine secretion, notably IFN-gamma secretion (as an example of a method of measuring IFN-gamma secretion, the Quantikine® immunoassay of R & D Systems Inc. (Catalog # CAIF00) may be used (Djoba Siawaya JF et al.)).

[0043] The present invention provides a recombinant EHV-1 comprising a DNA polymerase that has an asparagine (N) at amino acid position 752. DNA polymerase EHV-1 polypeptide homologues are understood to be within the scope of the present invention. invention. As used herein, the term "homologs" includes orthologs, analogs and paralogs. The tern "anologs" refers to two polynucleotides or polypeptides that have the same or function in foreign organisms. The term "ortholog" refers to two polynucleotides or polypeptides from different species but which evolved from a common ancestral gene by speciation. Orthologs typically encode polypeptides having the same or similar functions. The term "paralogs" refers to two polynucleotides or polypeptides that are related by duplication within a genome. Paralogs often have different functions, but these functions may be related. Analogs, orthologs and paralogs of a wild-type polypeptide may differ from the wild-type polypeptide through post-translational modifications, by differences in amino acid sequence, or by both. In particular, homologs of the invention generally exhibit at least 80-85%, 85-90%), 9095%), or 95%), 96%), 97%, 98%, 99% identity of sequence, with all or part of wild-type polypeptide or polynucleotide sequences, and exhibit similar function.

[0044] In one aspect of the present invention, the recombinant EHV-1 comprises an EHV-1 DNA polymerase, which comprises an asparagine (N) at amino acid position 752 or the position equivalent to a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5 %, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to SEQ ID NO: 2, 4, 6, 37, 13, 14, or 15 In another aspect, the present The invention provides fragments and variants of EHV-1 DNA polymerases, which can be readily prepared by one of ordinary skill in the art, using well-known molecular biology techniques. Variants are homologous polypeptides having an amino acid sequence of at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99 .3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical to the amino acid sequence as set forth in SEQ ID NO: 2, 4, 6 , 37, 13, 14, or 15. Variants include allelic variants. The term "allelic variant" refers to a polynucleotide or polypeptide containing polymorphisms that lead to changes in the amino acid sequences of a protein and that exists within a natural population (e.g., a virus species or variety). Such natural allelic variations can typically result in 1 to 5% variance of a polynucleotide or a polypeptide. Allelic variants can be identified by sequencing the nucleic acid sequence of interest in a number of different species, which can be easily accomplished using hybridization probes to identify the same genetic locus in those species. Any and all such nucleic acid and amino acid variations resulting from polymorphisms or variations which are the result of natural allelic variation and which do not alter the functional activity of the gene of interest are understood to be within the scope of the invention.

[0045] As used herein, the term "derivative" or "variant" refers to a polypeptide, or a nucleic acid encoding a polypeptide, that has one or more conservative amino acid variations or other minor modifications, such that ( 1) the corresponding polypeptide has a substantially equivalent function compared to the wild-type polypeptide or (2) an antibody raised against the polypeptide is immunoreactive with the wild-type polypeptide. These variants or derivatives include polypeptides with minor modifications of the EHV-1 polymerase DNA to primary amino acid sequences, which can result in peptides that have substantially equivalent activity compared to the corresponding unmodified polypeptide. Such modifications may be deliberate, as by site-directed mutagenesis, or they may be spontaneous. The term "variant" further contemplates deletions, additions and substitutions to the sequence, as long as the polypeptide functions to produce an immune response, as defined herein. Modifications can be any amino acid change at position 752 with the exception of SEQ ID NO amino acid positions: 2, 4, 6, 37, 13, 14, or 15.

[0046] The term "conservative variation" designates the substitution of an amino acid residue for another biologically similar residue, or the substitution of a nucleotide in a nucleic acid sequence in such a way that the encoded amino acid residue does not change or if another biologically similar residue. In this regard, particularly preferred substitutions will generally be conservative in nature, i.e., those substitutions that occur within a family of amino acids. For example, amino acids are generally divided into four families: (1) acidic, aspartate and glutamate; (2) BASIC-lysine, arginine, histidine; (3) non-polarimetry alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polarimetry glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan and tyrosine are sometimes classified as aromatic amino acids. Examples of conservative variations include the replacement of a hydrophobic residue such as isoleucine, valine, leucine or methionine with another hydrophobic residue, or the substitution of a polar residue for another polar residue, such as the substitution of arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine, and the like; or a similar conservative replacement of an amino acid with a structurally related amino acid, which will not have a major effect on biological activity. Proteins having substantially the same amino acid sequence as the reference molecule, but having small amino acid substitutions that do not substantially affect the immunogenicity of the protein are therefore within the definition of the reference polypeptide. All polypeptides produced by these modifications are included herein. The term "conservative variation" also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid provided that antibodies raised to the substituted polypeptide also immunoreact with the unsubstituted polypeptide.

[0047] Procedures for determining polypeptide and epitope fragments, such as generating libraries of overlapping peptides (Hemmer B. et al.), Pepscan (Geysen HM et al, 1984; HM Geysen et al, 1985, and Van der Zee R. et al; Geysen HM) and algorithms (De Groot A. et al; aro T. et al; Parker K. et al), can be used in the practice of the invention without undue experimentation. Generally, antibodies specifically bind a particular antigenic epitope. Specific non-limiting examples of epitopes include tetra to penta peptide sequence in a polypeptide, a tri-pentaerythritol glycoside sequence in a polysaccharide. In animals most antigens will present several or even many antigenic determinants simultaneously. Preferably, wherein the epitope is a protein fragment of a larger molecule that will have substantially the same immunological activity as the total protein.

[0048] In one aspect, the present invention provides a polynucleotide encoding an EHV-1 DNA polymerase, which comprises an asparagine (N) at amino acid position 752, or a position equivalent to a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5 %, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to SEQ ID NO: 2, 4, 6, 37, 13, 14, or 15, or a conservative variant thereof , an allelic variant, a homologue or an immunogenic fragment comprising at least eight, or at least ten consecutive amino acids of one of these polypeptides, or a combination of these polypeptides.

[0049] In another aspect, the present invention provides a polynucleotide having a nucleotide sequence as shown in SEQ ID NO: 1, 3, 5, or 36, or a variant thereof. In yet another aspect, the present invention provides a polynucleotide having at least 70%, 75%, 80%, 85%, 90%, 95%, 95%, 96%, 97%, 98%, 99%, 99.1 %, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9%) sequence identity to SEQ ID NO: 1, 3, 5 or 36, or a variant thereof.

[0050] The polynucleotides of the disclosure include sequences that are degenerate as a result of the genetic code, for example, the use of codons optimized for a specific host. As used herein, "optimized" refers to a polynucleotide that is genetically modified to increase its expression in a given species. To provide optimized coding polynucleotides for an EHV-1 DNA polymerase, the DNA sequence of the EHV-1 DNA polymerase gene can be modified to 1) comprise the codons preferred by highly expressed genes of a particular species; 2) comprises an A+T or content in the composition of nucleotide bases a which is substantially found in said species of G+C; 3) form an initiation sequence of said species; or 4) eliminates sequences that cause destabilization, inappropriate polyadenylation, degradation, and RNA extinction, or that form secondary structure hairpins or RNA splicing sites. The enhancement of EHV-1 DNA polymerase expression in said species can be achieved by utilizing the frequency of distribution codon usage in eukaryotes and prokaryotes, or in a particular species. The term "preferred codon usage frequency" refers to the preference exhibited by a specific host cell in using nucleotide codons to specify a given amino acid. There are 20 naturally occurring amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included in the description, while the DNA polymerase EHV-1 amino acid sequence encoded by the nucleotide sequence is functionally unchanged.

[0051] In one aspect, the present invention provides a recombinant EHV-1 that contains a mutant glycoprotein C (gC) gene. The term "mutant gC gene" refers to the EHV-1 gC gene, which is altered or engineered to result in a non-functional gC protein upon expression. The engineering or alteration of the gC gene includes mutation or deletion of a segment of the gC gene, which is essential for the expression of a functional gC protein. The gC gene deletion may be a deletion of the polynucleotides coding for the amino-terminal region of the gC protein. The amino-terminal deleted regions of the gC protein can be of any length, for example, a region of amino acids 1-10, or amino acids 11-20, or amino acids 21-30 or amino acids 31-40, or amino acids 41-60 or amino acids 6190, amino acids 91-120 or amino acids 121-160. The term "mutated gC gene" also includes the deletion of the entire gC gene from EHV-1, wherein the gC protein is not expressed.

[0052] In one aspect, the present invention provides an EHV-1 recombinant wherein the Glycoprotein C (gC) genes in the native (wild-type) EHV-1 genome encoding the GC protein is deleted. The term "Glycoprotein C (gC) gene" includes any gene or polynucleotide that encodes the glycoprotein C (gC) of EHV-1, and sapiens stems, fragments or variants thereof. The gC gene can encode a protein that has at least 75%, 80%, 85%, 90%, 95%, 95%, 96%, 97%, 98%, 99%, 99.1% of gC, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity of SEQ ID NO: 8, 10, or 12, or a variant thereof. The gC gene must have at least 75%, 80%, 85%, 90%, 95%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99 .3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%) or 99.9%) sequence identity to SEQ ID NO: 7, 9, or 11 is also encompassed by the present invention. In another aspect, the present invention provides an EHV-1 recombinant in which the glycoprotein C (gC) gene in the native (wild-type) EHV-1 genome encoding the protein is altered or engineered to result in a mutated protein. gC In yet another aspect, the engineered gC gene encodes a mutated gC protein in which the N-terminal region of the gC protein is deleted.

[0053] The sequence identity between two amino acid sequences can be established by the NCBI (National Center for Biotechnology Information) pairwise blast and the blosum62 matrix, using conventional parameters (see, for example, the BLAST or BLASTX algorithm is available at " National Center for Biotechnology Information" from the server (NCBI, Bethesda, Maryland., USA)), as well as in Altschul et al. ; and thus this document speaks using the BLAST algorithm or the or BLASTX and BLOSUM62 matrix by the term "blasts".

[0054] Alternatively, or in addition, the term "identity", for example with respect to a nucleotide or amino acid sequence, may indicate a quantitative measure of homology between two sequences. The percentage of sequence homology can be calculated as: (N re - N < 34 /) * 100 / N re , where N A / is the total number of non-identical residues in the two sequences when aligned and where N re is the number of residues in one of the sequences. Thus, the DNA sequence AGTCAGTC will have a 75% sequence identity with the sequence AATCAATC (N re = 8; v 7 = 2).

[0055] Alternatively, or in addition, "identity" with respect to sequences may refer to the number of positions with identical nucleotides or amino acids, divided by the number of nucleotides or amino acids in the shorter of the two sequences, where the alignment of the two sequences can be determined according to the algorithm of Wilbur and Lipman (Wilbur and Lipman), for example, using a window size of 20 nucleotides, a word length of 4 nucleotides, and a gap penalty of 4, and Computer-assisted analysis and interpretation of sequence data, including alignment, can be conveniently performed using commercially available programs (eg, Intelligenetics™ Suite, Intelligenetics Inc. CA). When RNA sequences are said to be similar, or have a degree of sequence identity or homology to the DNA sequences, it is the thymidine (T) in the DNA sequence that is considered equal to uracil (U) in the RNA sequence. Thus, RNA sequences are within the scope of the invention and can be derived from DNA sequences, for thymidine (T) in the DNA sequence is considered equal to uracil (U) in RNA sequences.

[0056] Sequence identity or sequence similarity between two amino acid sequences, or sequence identity between two nucleotide sequences can be determined using the Vector NTI software package (Invitrogen, 1600 Faraday Ave., Carlsbad, CA).

[0057] The recombinant vectors disclosed herein may include a polynucleotide that encodes a polypeptide, a variant thereof, or a fragment thereof. Recombinant vectors can include plasmids and viral vectors and can be used in vitro or for in vivo expression. Recombinant vectors may further include a signal peptide. Signal peptides are short peptide chains (3-60 amino acids in length) that direct the post-translational transport of a protein (which is synthesized in the cytoplasm) from certain organelles, such as the nucleus, mitochondrial matrix, endoplasmic reticulum, chloroplast , and apoplast peroxisome. Typically, naturally occurring, EHV-1 proteins can be translated as precursors, having an N-terminal signal peptide sequence and a "mature" protein domain. The signal peptide can be cleaved from the EHV-1 protein or a signal peptide from a secreted protein, for example the tissue plasminogen activator protein (tPA) signal peptide, in particular human tPA (S. Friezner Degen et al; R. Rickles et al; D. Berg, et al), or insulin-like growth factor 1 (IGF-1) signal peptide, in particular equine IGF1 (K. Otte et al), the Canine IGF1 (p Delafontaine et al), feline IGF1 (WO03/022886), bovine IGF-1 (S. Lien et al), porcine IGF-1 (M. Muller et al), chicken IGF-1 (Kajimoto Y. et al), IGF1 turkey (GenBank accession number AF074980). The signal peptide from IGF-1 can be natural or optimized which can be achieved by removing cryptic splicing sites and/or adapting the codon usage. After translation, the unprocessed polypeptide can be cleaved at a cleavage site to lead to the mature polypeptide. The cleavage site can be predicted using the method of Von Heijne (1986).

[0058] A plasmid may include a DNA transcription unit, for example, a nucleic acid sequence that allows it to replicate in a host cell, such as an origin of replication (prokaryotic or eukaryote). A plasmid may also include one or more selectable marker genes and other genetic elements known in the art. Circular and linear shapes of plasmids are encompassed herein.

[0059] In another aspect, the present invention relates to an in vivo expression vector, which comprises a polynucleotide sequence, which contains and expresses in vivo in a host the EHV-1 antigen, the polypeptides and/or variants. or its fragments.

[0060] The in vivo expression vector may include any transcriptional unit containing a polynucleotide or gene of interest and the essential elements for its expression in vivo. These expression vectors can be plasmids or recombinant viral vectors. For in vivo expression, the promoter may be of viral or cellular origin. In one embodiment, the promoter may be the cytomegalovirus (CMV), the early promoter (CMV-IE promoter), the SV40 virus early or late, or the Rous Sarcoma Virus LTR promoter, a promoter from a cytoskeleton, such as the desmin promoter ( Kwissa M. et al.), or the actin promoter (Miyazaki J. et al.). When several genes are present on the same plasmid, they may be provided in the same transcriptional unit or in different units.

[0061] As used herein, the term "plasmid" may include any DNA transcription unit comprising a polynucleotide according to the invention and the elements necessary for its expression in vivo in a cell or cells of the desired host or target. ; and, in this regard, it is noted that a supercoiled or non-supercoiled, circular plasmid, as well as a linear form, are intended to be within the scope of the invention. Plasmids may also comprise other transcriptional regulatory elements, such as, for example, intron-like stabilization sequences. In various embodiments, plasmids may include the first intron of CMV-IE (WO 89/01036), intron II of the rabbit beta-globin gene (van Ooyen et al), the signal sequence of the protein encoded by tissue plasminogen activator (tPA; Montgomery et al.), and/or a polyadenylation signal (poly A), in particular the bovine growth hormone (bGH) gene poly (US 5122458) or the rabbit poly A gene for beta- globin or SV40 virus.

[0062] Pharmaceutically acceptable carriers or diluents, or excipients or adjuvants for use are conventional. Remington's Pharmaceutical Sciences, by EW Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of the polypeptides, plasmids, viral vectors disclosed herein. In general, the nature of the vehicle or excipient will depend on the particular mode of administration being employed. For example, parenteral formulations generally comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a carrier. For solid compositions (e.g., lyophilisate, pellets, powder, pill, tablet, capsule or forms), conventional non-toxic solid carriers or diluents or excipients or adjuvants may include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers or diluents or excipients or adjuvants, immunogenic compositions to be administered may contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

[0063] Compositions or vaccines according to the invention may include recombinant vectors encoding any polypeptide or antigen according to the present invention, as described above.

[0064] Multiple inserts can be made to the same vector using different insert locations, or using the same insert location. When the same insertion site is used, each polynucleotide insert, which can be any aforementioned polynucleotide of the present invention, can be inserted under the control of the same and/or different promoters. Insertion can be done tail-to-tail, head-to-head, tail-to-head or head-to-tail. The IRES elements (Internal Ribosomes Site Entry, see EP0803573) can also be used to separate and to express multiple insertions operably linked to the same and/or different promoters.

[0065] More generally, the present invention encompasses, in in vivo expression vectors, including any plasmid (EP-A2 1001025, P. Chaudhuri) containing and expressing in vivo in a host the EHV-1 polypeptide polynucleotide or gene, variant or fragment as described above and the elements necessary for its expression in vivo.

[0066] In a specific, non-limiting example, plasmid pVR1020 or pVR1012 (VICAL Inc.; Lucas C. et al; Hartikka J. et al), pVR2001-TOPA (or pVR2001-TOPO) (Oliveira et al F.) or PABL 10 (US 6852705) can be used as a vector for inserting a polynucleotide sequence. The plasmid is derived from pVR1020 pVR1012 and contains the human tPA signal sequence. pVR1020 is a plasmid backbone available from Vical, Inc., (San Diego, CA), which has been previously used, see, for example, U.S. Patent Nos. 6,451, 769 and 7,078,507. As described in Oliveira et al, plasmid pVR2001-TOPO (or pVR2001-TOPA) is pVR1020 modified by the addition of topoisomerases flanking the cloning site and contain and express code for a secretory signal peptide, e.g. tissue plasminogen peptide activating signal (tPA), which increases the probability of producing a secreted protein (Oliveira F. et al).

[0067] Each plasmid may comprise or contain, or consist essentially of, the polynucleotide, according to the present invention, operably linked to a promoter or under the control of a promoter or dependent on a promoter, wherein the promoter may advantageously be adjacent to the polynucleotide for which expression is intended. In general, it is advantageous to employ a strong promoter that is functional in eukaryotic cells. An example of a useful promoter may be the cytomegalovirus immediate early promoter (CMV-IE) of human or murine origin, or may optionally be of another origin, such as rat or guinea pig. The CMV-IE promoter may comprise the actual part of the promoter, which may or may not be associated with the enhancer part. Reference may be made to EP 260 148, EP 323 597, US 5,168,062, 5,385,839, and 4,968,615, as well as WO 87/03905. The CMV-IE promoter can advantageously be a human CMV-IE (Boshart M. et al.) or a murine CMV-IE. In more general terms, the promoter can have either a viral or a cellular origin. A stronger viral promoter than other CMV-IE that can be usefully employed in the practice of the invention is the early/late promoter of the SV40 virus or the LTR promoter of the Rous sarcoma virus. A strong cellular promoter that can be usefully employed in the practice of the invention is the promoter of a cytoskeletal gene, such as the desmin promoter (Kwissa M. et al.), or the actin promoter (Miyazaki J. et al.) . Functional sub-fragments of these promoters, i.e. portions of these promoters which maintain appropriate promoter activity, are included within the scope of the present invention, for example the truncated CMV-IE promoters according to WO 98/00166 or US 6156567 and can be used in the practice of the invention. The promoter useful in the practice of the present invention, therefore, may include derivatives and/or sub-fragments of a full-length promoter that maintain the activity of the appropriate promoter and therefore function as a promoter, and which may advantageously have promoter activity that is substantially similar to that of the actual or full-length promoter from which the derived or secondary fragment is derived, e.g. similar to the activity of the truncated CMV-IE promoters of US 6156567 as compared to the activity of full-length CMV-IE promoters. Thus, a CMV-IE promoter in the practice of the invention may comprise or consist essentially of, or consist of the promoter portion of the full-length promoter and/or the enhancer portion of the full-length promoter, as well as derivatives and/or or sub-fragments.

[0068] Advantageously, the plasmids comprise or consist essentially of other expression control elements. It is especially advantageous to incorporate stabilizing sequence(s), e.g. the intron sequence(s), e.g. the first intron of hCMV-IE (WO 89/01036), intron II of the rabbit β-globin gene (van Ooyen et al.). As with the polyadenylation signal (poly A), for plasmids and viral vectors, other poxviruses, use may be made of the poly(A) signal from the bovine growth hormone (bGH) gene (see US 5122458), or the poly(A) signal from the rabbit globin gene - or the poly(A signal) from the SV40 virus.

[0069] More generally, the present invention encompasses, in in vivo expression vectors, including any recombinant viral vector that contains a polynucleotide or gene encoding one or more EHV-1 polypeptides, variants or fragments, as described above, including all the elements necessary for its expression in vivo.

[0070] The recombinant viral vector may be a herpesvirus, such as an Equine Herpesvirus-1 (EHV-1) as described above. The EHV-1 vector can be derived from the RacH strain, the RACL strain, the Ab4 strain, the V592 strain, the Kentucky D strain (TACC No. VR-700), the 438/77 strain (ATCC No. VR- 2229), the AB69 strain (ATCC No. VR-2581), the EHV-1 NY03 strain, or a combination of EHV-1 RacH RACL or strains. In one embodiment, the polynucleotide to be expressed is inserted under the control of a promoter functional in eukaryotic cells. Advantageously a CMV-IE promoter (murine or human). A poly(A) and terminator sequence can be inserted downstream of the polynucleotide to be expressed, for example, bovine growth hormone or a polyadenylation signal from the rabbit β-globin gene.

[0071] In one embodiment, the viral vector may be Newcastle Disease virus (US2012/052089). In another embodiment, the vial vector can be selected from, for example, poxviruses, especially avipox viruses, viruses such as fowlpox or canarypox viruses. In one embodiment, the fowlpox virus is a TROVAC (see WO 96/40241). In another embodiment, the vector is an ALVAC canarypox. The use of these recombinant viral vectors and the insertion of polynucleotides or genes of interest are fully described in US 5174993; US 5,505,941 and US 5,766,599 for fowlpox, and in US 5,756,103 for canaripox. More than one insertion site within the viral genome can be used for the insertion of multiple genes of interest.

[0072] For recombinant vectors based on a poxvirus vector, a vaccinia virus or an attenuated vaccinia virus, (e.g. MVA, a modified Ankara strain obtained after more than 570 passages of the Ankara vaccine strain in fibroblasts chick embryo; see Stickl & Hochstein-Mintzel, Sutter et al; available as ATCC VR-1508, or NYVAC, see US 5494807 and US Patent No. 5494807 which discusses NYVAC construction as well as NYVAC variations with Additional ORFs excluded from the Copenhagen vaccinia virus strain genome, as well as the insertion of heterologous encoding nucleic acid molecules at sites of this recombinant, and also, the use of combine promoters; see also WO 96/40241), a virus or an avipox virus attenuated avipox (e.g., canarypox, fowlpox, dovepox, pigeonpox, quailpox, ALVAC or TROVAC; see, for example, US Patent Nos. 5505941, 5494807) can be used. Attenuated canarypox viruses are described in US 5756103 (ALVAC) and WO 01/05934. Reference is also made to US5,766,599 which belongs to the attenuated TROVAC fowlpox strain. Reference is made to canaripox available from the ATCC under accession number VR-111. Several vaccine strains of the avian yaws virus are also available, for example the DIFTOSEC CT strain marketed by Merial and the NOBILIS variole vaccine marketed by EFP INTER. For information on the method used to generate its recombinants and how to administer its recombinants, the skilled person can refer to the documents herein and cited WO 90/12882, for example, as for the vaccinia virus, mention is made of US Patent Nos. 4,769,330, 4,722,848, 4,603,112, 5,110,587, 5,494,807, and 5,762,938 inter alia; as, mention is made of US Patent Nos. 5,174,993, 5,505,941 and 5,766,599 namely; such as canarypox, reference is made to US Patent No. 5,756,103, inter alia. When the expression vector is a vaccinia virus, the insertion site or sites of the polynucleotide or polynucleotides to be expressed are advantageously in the thymidine kinase gene or insertion site (TK), the hemagglutinin (HA) or gene insertion site, which encodes the type A inclusion body region (ATI). In the case of canarypoxviruses, advantageously the insertion site or sites are ORF(s) C3, C5 and/or C6. In the case of fowlpox, advantageously the insertion site or sites are ORF F7 and/or F8. The MVA virus insertion site or sites are advantageously as in several publications, including Carroll MW et al; Stittelaar KJ et al; Sutter G. et al; and, in this regard, it is also worth noting that the complete genome of MVA is described in Antoine G., Virology, which allows the person skilled in the art to use other insertion sites or other promoters. Advantageously, the polynucleotide to be expressed is inserted under the control of a poxvirus-specific promoter, for example, the 7.5 kDa vaccinia promoter (Cochran et al.), the I3L vaccine promoter (Riviere et al. al.), the vaccinia HA promoter (Shida), the cowpox virus ATI promoter (Funahashi et al.), the vaccinia H6 promoter (Taylor J. et al; Guo P. et al J.; Perkus M. et al.)

[0073] Any of the polynucleotides described herein can be expressed in vitro by transferring DNA or expression vectors into a suitable host cell. The host cell may be prokaryotic or eukaryotic. The term "host cell" also includes any progeny of the subject host cell. Stable transfer methods, which means that the foreign polynucleotide is continuously maintained in the host cell, are known in the art. Host cells can include bacteria (e.g., Escherichia coli), yeast, insect cells, and vertebrate cells. Methods of expressing DNA sequences in eukaryotic cells are well known in the art. As a method for in vivo expression, recombinant baculovirus vectors (e.g., Autographa California Nuclear Polyhedrosis Virus (AcNPV)) can be used with the nucleic acids disclosed herein. For example, polyhedrin promoters can be used with insect cells (e.g. Spodoptera frugiperda cells, such as Sf9 cells available from the ATCC under accession number CRL 1711, or Sf21 cells) (see, for example, Smith et al; Pennock et al; Vialard et al; Verne one.; O'Reilly et al.; Kidd l M. & Emery VC; EP 0370573; EP 0265785, US 4745051). For expression, the starter BaculoGold package (Cat # 21001 K) from Pharmingen (Becton Dickinson) can be used. As a method for recombinant expression of E. coli it can be used with a vector. For example, when cloning in bacterial systems, inducible promoters such as the arabinose promoter, bacteriophage lambda PL, plac, ptrp, ptac (ptrp-lac hybrid promoter), and the like can be used. Transformation of a host cell with recombinant DNA can be accomplished by conventional techniques that are well known to those skilled in the art. When the host is prokaryotic, such as E. coli, competent cells that are capable of uptake of DNA can be prepared from cells harvested after the exponential growth phase and subsequently treated by the CaCl2 method, using procedures well known in the art. . Alternatively, MgCl2 or RbCl can be used. Transformation can also be carried out by electroporation. When the host is a eukaryote, such DNA transduction methods as calcium phosphate coprecipitates can be used conventional mechanical procedures, such as microinjection, electroporation, insertion of a plasmid enclosed in liposomes or viral vectors. Eukaryotic cells can also be co-transformed with polynucleotide sequences from L. Iongipalpis, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene. Another method is to use a eukaryotic viral vector (see above), such as a herpes virus or adenovirus (e.g. canine adenovirus 2), to transiently transduce eukaryotic cells and express the protein (Gluzman EA). In addition, a transfection agent such as dioleoyl-phosphatidyl-ethanolamme (DOPE) may be used.

[0074] Isolation and purification of the recombinantly expressed polypeptide can be performed by conventional means, including preparative chromatography (e.g., size exclusion, ion exchange, affinity), selective precipitation, and ultrafiltration. State-of-the-art examples of techniques that can be used, but not limited to, can be found in "Protein Purification Applications", second edition, edited by Simon Roe and available from Oxford University Press. This expressed recombinant polypeptide is part of the present disclosure. Methods for producing any polypeptide according to the present invention, as described above, also encompass, in particular, the use of a recombinant expression vector comprising a polynucleotide, according to the disclosure, and a host cell.

[0075] The vaccines of recombinant viral vectors that contain according to the invention can be lyophilized, advantageously, with a stabilizer. Lyophilization can be carried out according to well known conventional freeze-drying procedures. Pharmaceutically acceptable or veterinary stabilizers can be carbohydrates (e.g. sorbitol, mannitol, lactose, sucrose, glucose, dextran, trehalose), sodium glutamate (Tsvetkov T et al; Israeli E et al), proteins such as peptone , albumin, lactalbumin or casein, protein-containing agents such as skim milk (Mills CK et al; Wolff E et al), and buffers (eg phosphate buffer, alkali metal phosphate buffer). An adjuvant can be used to make lyophilized soluble preparations.

[0076] Any vaccine composition according to the invention may advantageously also contain one or more adjuvants.

[0077] Plasmid-based vaccines can be formulated with cationic lipids, advantageously with DMRIE (N-(2-hydroxyethyl)N,N-dimethyl-2,3-bis(tetradecyloxy)-propanammonium; WO96/34109) and advantageously , in association with a neutral lipid, for example DOPE (dioleoyl-phosphatidyl-ethanolamine; Behr JP), to form DMRIE-DOPE. In one embodiment, mixing is done extemporaneously, and prior to administration, it is advantageous to wait about 10 min to about 60 minutes, for example about 30 min, for proper mixing. When DOPE is used, the molar ratio of DMRIE/DOPE can be from 95/5 to 5/95 and 1/1 is advantageously. The weight ratio of plasmid/DMRIE or DMRIE-DOPE adjuvant is, for example, from 50/1 to 1/10, from 10/1 to 1/5 or from 1/1 to 1/2.

[0078] Optionally, the cytokine can be added to the composition, especially GM-CSF or induce Thl cytokines (eg IL-12). These cytokines can be added to the composition in the form of a plasmid encoding the cytokine protein. In one embodiment, the cytokines are of canine origin, for example, canine GM-CSF gene sequence that has been deposited in the GenBank database (accession number S49738). This sequence can be used to create said plasmid in a similar way to what was done in WO 00/77210.

insaturado radicais que têm pelo menos dois átomos de carbono. Por exemplo, os radicais são aqueles que contêm de 2 a 4 átomos de carbono, por exemplo, vinil allyls e outros grupos etilenicamente insaturados. Os radicais insaturados podem eles mesmos conter outros substituintes, tal como metilo. Os produtos vendidos sob o nome CARBOPOL® (BF Goodrich, Ohio, EUA) são adequados. Os produtos são reticulados com uma alil-sacarose ou com alil- pentaeritritol. Entre eles, pode ser vantajosamente mencionado CARBOPOL® 974P, 934P e 97 IP.[0079] Recombinant viral vector-based vaccine can be combined with fMLP (N-formyl-methionyl-leucyl-phenylalanine; US 6017537) and/or Carbomer adjuvant (Phameuropa Vol 8, No. 2, June 1996) . Those skilled in the art may also refer to US 2909462 which describes such acrylic polymers cross-linked with a polyhydroxy compound having at least 3 hydroxyl groups, advantageously not more than 8, the hydrogen atoms of at least three groups hydroxyl being replaced by aliphatic

unsaturated radicals that have at least two carbon atoms. For example, radicals are those containing from 2 to 4 carbon atoms, for example vinyl allyls and other ethylenically unsaturated groups. Unsaturated radicals may themselves contain other substituents, such as methyl. Products sold under the name CARBOPOL® (BF Goodrich, Ohio, USA) are suitable. The products are cross-linked with an allyl sucrose or with allyl pentaerythritol. Among them, CARBOPOL® 974P, 934P and 97 IP can be advantageously mentioned.

[0080] Among the copolymers of maleic anhydride and alkenyl derivative, the copolymers EMA® (Monsanto) which are copolymers of maleic anhydride and ethylene, linear or cross-linked, for example, cross-linked with divinyl ether, are advantageous. Reference may be made to J. Fields et al.

[0081] Acrylic or methacrylic acid polymers and EMA® copolymers are formed, for example, from base units of the following formula in which: R1 and R2, which are identical or different, represent H or CH3 x = 0 or 1, preferably x = 1 y = 1 or 2, with x + y = 2 For EMA® copolymers, x = 0 and y = 2 For carbomers, x = y = 1.

[0082] The dissolution of these polymers in water leads to an acid solution, which is advantageously neutralized at a physiological pH, so as to provide the adjuvant solution in which the vaccine itself is constituted. The carboxyl groups of the polymer are in part then COO" form.

[0083] In one embodiment, a solution of adjuvant, especially carbomer (Pharmeuropa vol. 8, No. 2, June 1996), is prepared in distilled water, advantageously in the presence of sodium chloride, the solution being obtained in an acidic pH. This stock solution is diluted by adding the desired amount (to obtain the desired final concentration), or a substantial part thereof, of water loaded with NaCl, advantageously saline solution (NaCl 9 g / 1) all at once, in several portions with concomitant or subsequent neutralization (pH 7.3-7.4), advantageously with NaOH. This solution, at physiological pH, is used to mix with the vaccine, which can be specially stored in lyophilized, liquid or frozen form.

[0084] The concentration of the polymer in the final vaccine composition can be from 0.01% to 2% w/v, 0.06-1% w/v, or from 0.1 to 0.6% w/v.

[0085] The subunit vaccine can be combined with adjuvants such as water-in-oil, water-in-oil-in-water emulsions based on mineral oil and/or vegetable oil and non-ionic surfactants such as block copolymers , TWEEN, SPAN®. Such emulsions are namely those described on page 147 of "Vaccine design - A Subunit and Adjuvant Approach", Pharmaceutical Biotechnology, 1995, or TS emulsions, namely the TS6 emulsion, and the emulsions, namely LF emulsion LF2 (for both emulsions and TS LF, see WO 04/024027). Other suitable adjuvants are, for example, vitamin E, saponins, and CARBOPOL® (Noveon, see WO 99/51269, WO 99/44633), aluminum hydroxide or aluminum phosphate ("Vaccine Design, subunit and adjuvant approach" , Pharmaceutical Biotechnology, vol. 6, 1995), biological adjuvants (i.e., C4b, namely murine C4b (Ogata RT., et al) or equine C4b, GM-CSF, in particular equine GM-CSF (US 6645740)) , toxins (e.g. CTA or CTB cholera toxins, Escherichia coli, the heat-labile toxins or LTA LTB (Olsen et al CW; Fingerut E et al; Zurbriggen R et al Peppoloni S. et al), and CpG (or that is, CpG # 2395 (see Jurk M et al), CpG # 2142 (see SEQ ID NO: 890 in EP 1 221 955).

[0086] The composition or vaccine may also be associated with at least one EHV-1 antigen, eg inactivated EHV-1. In a particular embodiment, the EHV-1 strain may be the RacH strain, the RACL strain, the Ab4 strain, the V592 strain, the Kentucky D strain (TACC No. VR-700), the 438/77 strain (ATCC No. VR-2229), AB69 strain (ATCC No. VR-2581), EHV-1 NY03, or a combination of EHV-1 RacH RACL or strains. These strains of EHV-1 can be inactivated by chemical or physical methods. Chemical methods are notably GLP, formaldehyde. Physical methods may be, in particular, ultrasound. The inactivated EHV-1 from the vaccine can be combined with adjuvants such as those described above for subunit vaccines.

[0087] Another aspect of the present invention pertains to methods of vaccinating a host against EHV-1 using the vaccines or compositions disclosed herein.

[0088] The host can be any animal. In one embodiment, the host is a horse.

[0089] The routes of administration can be, for example, intramuscularly (IM) or intradermally (ID) or transdermally (TD) or subcutaneously (SC). The means of administration may be, for example, a syringe with a needle, or a needle-free device, or a syringe with a needle attached to the electrotransfer (ET) treatment, or a needle-free device attached to the ET treatment.

[0090] In the case of plasmid-based vaccines, advantageous routes of administration can be ID or IM. This administration may be through the use of a syringe with a needle or with a needle-free device such as Dermojet or Biojector (Bioject, Oregon, USA) or Vetjet™ (Merial) or Vitajet™ (Bioject Inc.), see US 2006/ 0034867. The dosage can be from 50 µg to 500 µg per plasmid. When DMRIE-DOPE is added, 100 µg per plasmid can be used. When GM-CSF or other cytokines are used, the plasmid encoding the protein can be present at a dosage of between about 200 µg to about 500 mg and can be 200 µg. The dose volume may be between 0.01 ml and 0.5 ml, for example 0.25 ml. Administration can be provided with multiple injection points.

[0091] Alternatively, plasmid-based vaccines can be administered via IM coupled to electrotransfer (ET) treatment. ET treatment can be performed using an electrotransfer apparatus and manufacturer's specifications (i.e. Sphergen G250 generator (Sphergen SARL, Evry Genopole, France); DNA MedPulser® electroporation system (Innovio Biomedical Corporation, San Diego, California, USA)).

[0092] In the case of vaccines based on recombinant viral vectors, the routes of administration can advantageously be SC, IM, TD, or ID. This administration can be done by a syringe with a needle or with a needle free device such as Dermojet or Biojector (Bioject, Oregon, USA) or Vetjet™ (Merial) or Vitajet™ (Bioject Inc.). The dosage can be from about 10 4 pfu to about 10 9 cfu per recombinant EHV vector. The dose volume can be from about 0.01 ml to 0.2 ml, and is advantageously 0.1 ml. Administration may comprise various injection points.

[0093] By the I.M. route, the volume of vaccine can be supplied to 0.2-2 ml, in particular from about 0.5 to 1 ml. The same dosages are used for any of the vectors of the present invention.

[0094] In the case of subunit vaccines, the route of administration may advantageously be via SC or IM or TD or ID. This administration can be done by a syringe with a needle or with a needle free device such as Dermojet or Biojector (Bioject, Oregon, USA) or Vetjet™ (Merial) or Vitajet™ (Bioject Inc.). The dosage may be from about 50 to about 500 mg, in particular from about 50 to about 150 mg, and more particularly from about 50 to about 100 µg. The volume of sub-unit vaccine is given 0.2-2 ml, in particular from about 0.5 to 1 ml.

[0095] In one aspect, the present invention relates to a vaccine strategy, which is based on a prime-boost administration regimen, where prime administration and booster administration utilize a composition comprising a pharmaceutically acceptable carrier, excipient or veterinary, diluent, adjuvant, or vehicle and the EHV-1 recombinant of the present invention. In another aspect, the present invention relates to a method of vaccinating an EHV-1 susceptible subject or host, which comprises a preferred booster administration regimen.

[0096] A prime-boost regimen comprises at least one prime-administration and at least one boost-administration via at least one antigen and/or its variants or its common fragments. The vaccine used in the primary administration may be of a different nature from those used as a later booster vaccine. Note further that both the prime and administration may comprise the recombinant EHV-1 boost of the present invention. The PriME-boost administration may comprise one or more administrations. Likewise, the impulse-administration may comprise one or more administrations.

[0097] Routes of administration, doses and volumes are as previously described herein.

[0098] Prime-boost administrations can be performed 2 to 6 weeks apart, eg about 4 weeks apart. According to one embodiment, a semi-annual booster dose or an annual booster is also provided.

[0099] In one embodiment, the prime-boost administration regimen comprises at least one first-administration of a recombinant EHV-1 vaccine, or composition of the present invention, and at least one booster administration of a viral vaccine. inactivated, comprising the EHV-1 antigen, or a plasmid-based vaccine expressing the EHV-1 antigen, or a subunit vaccine comprising the EHV-1 antigen, or a combination thereof.

[0100] In another embodiment, the prime-boost administration regimen comprises at least one first administration of an inactivated virus vaccine comprising the EHV-1 antigen, or of a plasmid-based vaccine expressing the EHV antigen -1, or a subunit vaccine comprising the EHV-1 antigen, or a combination thereof, and at least one booster administration of a recombinant EHV-1 vaccine or composition of the present invention.

[0101] Another aspect of the present invention relates to a kit for inducing and boosting vaccination in accordance with the present invention. The kit may comprise at least two tubes of: a first vial containing a vaccine for primary vaccination according to the present invention and a second vial containing a vaccine for booster vaccination according to the present invention. The kit may advantageously contain additional first or second vials for additional main vaccinations or additional booster vaccinations.

[0102] In one embodiment, the kit may comprise two vials, one containing a plasmid-based vaccine for the primary vaccination in accordance with the present invention, the other vial containing a recombinant viral vector-based vaccine for the vaccination of reinforcement according to the present invention.

[0103] The invention will now be further described by way of the following non-limiting examples.

EXAMPLES

[0104] Without further elaboration, it is believed that one skilled in the art can, using the foregoing descriptions, practice the present invention to its fullest extent. The following detailed examples are to be construed as illustrative only, and not limitations of the foregoing disclosure in any way. Those skilled in the art will readily recognize appropriate procedural variations in both reagents and reaction conditions and techniques.

[0105] The construction of DNA inserts, plasmids and recombinant viral vectors was performed using conventional molecular biology techniques described by J. Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989). All restriction fragments used for the present invention were isolated using the "Geneclean" kit (Bio 101 Inc., La Jolla, Calif.). Example 1 - Construction of the cell line RK13_Pol Cells and Viruses

[0106] Rabbit kidney cell (RK13) was maintained in Earle's minimal essential medium (EMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin and 0.1 mg/ml of streptomycin (1% Pen/Strep). Equine skin fibroblast cell line (NBL6) was maintained in EMEM supplemented with 10% FBS, 29 mg/ml L-glutamate, 1% Pen/Strep and 1% non-essential amino acids (Gibco BRL). EHV-1 strains and RacLl 1 were grown in fresh NY03 R 13 cells. NY03, harboring a non-neurological form of DNA polymerase (N752 Pol), was isolated from an aborted foal from a New York farm in 2003. Construction of the R 13_Pol cell line

[0107] To support the growth of a Pol-negative RacLl mutant, a rabbit kidney cell line designated RK13_Pol expressing the non-neurological form of the polymerase (N752) was first generated. The entire NY03 polymerase open reading frame was amplified from viral genomic DNA, which was extracted from virus-infected R13 cells. The polymerase chain reaction (PCR) was performed using AccuPrime DNA polymerase (Invitrogen, Carlsbad, CA, USA) and a PN1/PN2 primer pair (Table 1). The amplified fragment, flanked with a Hind III restriction site at the 5' end and a BamHI site, as well as an HA tag at the 3' end, was cloned into plasmid pCDNA3 to generate pCDNA3-Pol. After digestion with Pvu I, 4 µg of recombinant plasmid pCDNA3-Pol was linearized and transfected into R13 cells grown in six-well plates using Lipofectamine 2000 (Invitrogen). Transfected cells were propagated in EMEM containing 10% FBS and 1.2 mg/ml G418 (Merck). A single cell clone in which each cell was shown to express DNA polymerase by indirect immunofluorescence (IF A) reaction was selected and named R 13_Pol. By IFA using anti-HA MAb, expression of the polymerase in R 13_Pol could be demonstrated (Figure 3) and it was shown to be stable in ten fold passages. Table 1 Sequences of primers.

Example 2 - Plasmid and viral mutagenesis Mutant virus generation

[0108] Conventional homologous recombination strategy was employed for all genetic manipulations. Two 1.7kbp fragments flanking each side of DNA polymerase were amplified using thermostable Pfu polymerase (Promega) and P1/P2 and P3/P4 primer pairs from RacLl a genome. Another two pairs of primers P5/P6 and P7/P8 were used to amplify the HCMV (human cytomegalovirus) promoter and gene (enhanced green fluorescent protein) from the EYFP plasmid pEYFP-N1, separately. The 5' ends of primers P2 and P5, P8 and P3, as well as P6 and P7 carry homologous 21-23bp sequences. With a PCR overlay using P5 and P8, the HCMV promoter gene and EYFP were fused and cloned into plasmid pCR2.1-TOPO (Invitrogen), resulting in pCES1. For the transport construction for the pCR-Topo-PlP4 plasmid, the above two homologous arms and the EYFP express cassette in pCES1 were combined by PCR overlay using the P1/P4 primers and cloned into pCR2.1-Topo. Using pCES1 as a template and primer pair P9/P10, EYFP cassette flanked with SacII and NheI restriction sites as well as upstream and downstream loxP sequences was amplified and cloned into pCR2.1-Topo resulting in plasmid pCES2. To generate the plasmid pCR-Topo-gC, a fragment containing the 5.87kbp gC, which was amplified from a RacL1 genome using primers gC-1 and gC-2, was cloned into pCR2.1-Topo. After digestion with SacII and NheI, EYFP cassette was released from pCES2 and transferred to PCR-Topo-GC to replace gC gene and the recombinant shuttle plasmid was named Topo-AgC-EYFP. Plasmids pCESl, pCES2, PCR-Topo-PlP4 and Topo-AGC-EYFP were identified by digestion and sequencing. The cloning scheme is illustrated in Figure 2.

[0109] For the DNA polymerase mutation, 1 μg of RacLl viral DNA and 10 g of plasmid pCR-Topo-PlP4 were co-transfected into RK13_Pol cells in a 6-well plate by calcium phosphate precipitation. Two days after transfection, green plaques were observed under a fluorescence microscope (Axiovert 25, Zeiss). Whole virus was harvested after 2 freeze-thaw cycles and grown in fresh R 13_Pol cells that were covered with 1.5% methylcellulose in 2% EMEM-FBS at 1 hour post-infection. After three rounds of purification, a homogenous Pol-negative RacLl mutant, named LI EYFP, was generated and identified using restriction fragment size polymorphism analysis (RFLPs). To restore N752 Pol, a 7kbp fragment, termed pi p4pol, was amplified from the EHV-1 NY03 genome using AccuPrime Tag polymerase and the P1/P4 primers. By co-transfecting 1 µg of L1 EYFP viral DNA and 4 µg of PCR product into plp4pol R 13 cells, homologous recombination was again used to introduce variant N752 Pol and RacL1 a mutant LI 1_D752N was obtained. The D752 mutation in N752 polymerase was confirmed (Fig. 2D) by sequencing a 780bp Pol fragment that was amplified from L11 D752N using the sequencing primers and poly1 poly2.

[0110] Next steps were to exclude open reading gC from L1 1_D752N mutant. Briefly, 1 µg of the L1 D752N viral DNA frame and 10 µg of Topo-AGC-EYFP plasmid transfer were co-transfected into RK13 cells. The SacII/NheI fragment (1.2kbp in length) in the gC gene was replaced with an EYFP cassette leading to the generation of a recombinant L1 D752N AgC EYFP virus. The EYFP selection marker was excised by expressing the Cre recombinase by co-transfecting L11_D752N AgC EYFP and plasmid pCAGGS-NLS/Cre into RK13 cells. White plaques were purified and a GC-negative, non-neurological RacLl mutant LI 1 D752N AgC was finally engineered. By homologous recombination between the viral DNA LI 1 D752N AgC EYFP and plasmid pCR-Topo-gC, the gC gene was repaired, resulting in a reverting gC LI 1_D752N AgC rev (Fig.2). The gC gene deletion and the absence of gC protein were confirmed by PCR using primer pair identification of gC-l/2 and gC-AGC-l/AGC-2, sequencing, as well as indirect immunofluorescence (IFA) assays. .

[0111] All primers used for plasmid construction and sequencing were collected from Table 1. Indirect Immunofluorescence Assay (IFA)

[0112] For the detection of DNA polymerase, which was expressed in R 13_Pol cells, the monoclonal antibody (MAb) directed against the HA tag (H3663, Sigma) was used. Cells grown in R 13_Pol a 6-well plate were washed with phosphate-buffered saline (PBS) and fixed in 3.5% paraformaldehyde in PBS for 1 h at room temperature (RT), followed by a 5 minute incubation in PBS. containing 30 mM glycine and another 5min permeation in 0.1% Triton X-100 in PBS. After washing with PBS, cells were blocked with PBS-3% bovine serum albumin (BSA) for 30 min at RT. Cells were then incubated with the primary antibody at a 1:10,000 dilution in PBS-3% BSA for 1 h at room temperature and washed extensively with PBS. Secondary antibody (Alexa Fluor568-conjugated goat anti-mouse IgG, Invitrogen) was added at a 1:2000 dilution in PBS-3%>BSA and incubated for 1 h at room temperature. After thorough washing to 3 times of lómin, fluorescence signal was inspected under the inverted fluorescence microscope (Axiovert 25, Zeiss).

[0113] To confirm the absence of gC protein, R13 cells were seeded in a 6-well plate and infected with wild type RacLl l, mutant LI 1_D752N AgC or reverting gC LI 1_D752N AgC rev at a multiplicity of infection (MOI ) of 0.0001. One hour later, the viruses were removed and the infected cells were covered with 1.5% methylcellulose in 2% EMEM-FBS. After 48h incubation at 37°C, cells were fixed and blocked as described above. The mAb 1G4 against EHV-1 and gC protein B8 against EHV-1 of protein gM were used as the primary antibodies at a dilution of 1:100 and 1:200, separately. After incubation with secondary antibody (Alexa Fluor568-conjugated goat anti-mouse IgG) to 1h and cells were washed and plaques were observed. Characterization of virus mutants

[0114] A Pol-negative L1 EYFP virus mutant was generated by replacing the polymerase gene with authentic EYFP via co-transfection of RacL1 1 viral DNA and transport to the PCR-Topo-PlP4 plasmid in RK13_Pol. LI EYFP was shown to be able to grow only on RK13_Pol but not on non-complement R 13 cells (Fig.4), which confirms that DNA polymerase is essential for the growth of EHV-1 virus in vitro. Mutation from neurological D752 to non-neurological N752 genotype was then achieved by restoring the non-neurological EHV-1 NY03 polymerase gene into LI EYFP. The single amino acid variation was confirmed by sequencing a 780bp amplicon from the LI 1 mutant D752N. Based on 1 L1 D752N, the SacII/NheI region representing a 1222bp fragment (from nucleotide position 94-1315) in gC gene was replaced with EYFP, resulting in a GC-negative L11_D752N AgC EYFP intermediate. By Cre expression, the EYFP cassette was finally removed, leaving a copy of the LoxP sequence (34bp) between the SacII and NheI restriction sites in the designed L1_D752N AgC mutant. To confirm the deletion of the gC gene, PCR was performed. Although from the parental mutant LI 1_D752N, 5.87kbp 1.6kbp and fragments can be amplified using the primer pair of gC-1/2 and gC-AGC-1/AGC-2, from LI 1_D752N AgC, however, 4.68kbp and 410bp fragments were amplified (Fig.2), suggesting that 1.2kbp deletion in the gC gene was present. By IFA using anti-EHV-1 gC MAb, it could be shown that the gC protein was detectable in cells infected with RacL1 1 or the revertant LI 1_D752N AgC rev, but in cells infected with LI 1_D752N AgC, the protein expression was gC abolished (Fig.5). RacLl a GC-negative and non-neurological mutant was thus successfully generated. Example 3 - Characterization of virus growth IN VITRO Virus attachment assay

[0115] Virus attachment assay was performed as described in Sun et al. (J Gen Virol 77, 493-500, 1996) with slight modifications. Monolayers of R 13 cells seeded in 6-well plates were cooled for 1 h at 4°C and infected with RacL1 1, L1 1_D752N, L1 or L1 1_D752N AgC 1_D752N AgC rev with an MOI of 400 plaque forming units (PFU) per pit. Viruses were allowed to bind for various lengths of time (0, 15, 30, 60, 120, 240 min) at 4°C. At various time points, infected cells were washed three times with PBS and covered with EMEM containing 2% FBS and 1.5% methylcellulose. After incubation for 3 days at 37°C, cells were fixed with 10% formaldehyde and stained with 0.3% crystal violet. Plaques were counted and the percentage of virus binding at each time point was calculated, relative to the 240 min time point, which was defined as 100% binding. Plate size and growth kinetics in one step

[0116] To compare plate sizes, RK13 cells grown in 6-well plates were infected with wild-type RacL1 1 and mutants LI 1_D752N, LI or LI 1_D752N AGC 1_D752N AgC rev at an MOI of 0.0001 and covered with 1.5% methyleellulose in EMEM containing 2% FBS at 2hpi, post infection Three days, plaques were visualized by IFA using anti-EHV-1 B8 gM MAb. For each virus, 50 plaques were photographed and the mean plaque areas were measured using imaging software (http://rsb.info.nih.gov/ij/). RacLl 1-induced plaque sizes were set to 100%. Mean percentage and standard deviation were calculated from three independent experiments. For single-step growth kinetics assays, RK13 cells seeded in 24 plates were infected at an MOI of 3 Viruses were allowed to bind for 1 hour at 4°C followed by a 1.5 h penetration step at 37°C. After washing twice with PBS, infected cells were treated with ice citrate-buffered saline (CBS, pH 3.0) for 3 minutes to remove surface virus. At different time points (0, 4, 8, 12, 24, 36, 48hpi), supernatants and cells were collected separately. Extracellular and cell-associated viral titers were determined using standard plaque assays. One-step growth curves were calculated from three independent experiments.

In vitro growth properties

[0117] The properties of various virus mutants in in vitro growth cell cultures were analyzed. To investigate the impact of gC deletion on the ability of L11 D752N AgC to binding target cells, an attachment assay was performed. As expected, LI 1_D752N AgC was shown to bind less efficiently on RK13 cells when compared to either wild type RacL1 1, parental LI 1_D752N virus or the revertant LI 1_D752N AgC rev (Fig.6). The ability of L11_D752N AgC to spread from cell to cell was determined by comparing plate sizes. Considering that any significant difference between wild type, parental virus and reverting gC could be observed, in the area of the plaque in relation formed by LI a D752N AgC, however, it was 10% higher (<0.0001) than those of the others (figure 7), which indicates that, in the absence of gC, cell-to-cell spread of EHV-1 is even more efficient. With regard to growth kinetics, in vitro replication of LI 1_D752N AgC was less effective, as demonstrated by extracellular and intracellular titers that were reduced by approximately 20-fold and 10-fold, respectively, relative to wild-type, parental virus. or reverting gC (Fig. 8). Viral titers in culture supernatants of cells infected with wild type RacL1 1, parental 1_D752N LI virus or repair L1 1_D752N AgC rev reached intracellular titers at about 12 hours post-infection. In cells infected with LI 1 D752N AGC, however, a delayed release of infectious progeny was observed whereby the extracellular titer crossed the intracellular titer at 16-18 hours post-infection. From these results, it can be concluded that the in vitro growth of LI 1_D752N AgC was impaired in virus binding and egress although it could form slightly larger plaques.

Example 4 - Rat Vaccination

[0118] Wild type RacL1 1 as well as L11_D752N, L11_D752N AGC and L11_D752N AGC rev mutants were purified by ultracentrifugation to 1h at 27,000rpm. The pellet was suspended in PBS, titrated on fresh RK13 cells, aliquoted and stored at -70°C until use. Three-week-old female BALB/c mice (Harlan) were randomly assigned to five groups of 16 mice each and allowed to adapt to one another for one week at the Biosafety Level-2 facility. On day 0, all mice were weighed, anesthetized with O. LML / log xylazine / ketamine and four groups were inoculated intranasally (IN) with each of the viruses at a dose of 1 x 10 5 PFU in 20μI - of PBS. The last group was used as a negative control and received 20 µg of PBS. Body weight of each mouse was inspected until day 14 post-inoculation (pi). Three mice from each group were sacrificed on day 2 and on day 4, the lung was removed, part of which was homogenized and used for determination of virus titers by standard titration on R 13 cells, and the rest was fixed with formaldehyde. at 10% and processed for histopathological analysis. On day 28, mice were challenged with wild type RacL1 1 at a dose of 1 x 10 5 PFU intranasally. Data on individual body weights were collected over two weeks. On day 2 and day 4 after challenge (pc), three mice from each group were sacrificed to remove the lung. Virus titers in lung tissues were determined in fresh RK13 cells after homogenization.

[0119] On day 2 pi, it means virus titers in lungs infected with RacLl 1, LI 1 D752N or LI 1_D752N AgC rev reached 5366 PFU/mg, 3450 PFU/mg and 4800 PFU/mg respectively (Fig.9) , among which no statistically significant difference was observed (p>0.442). This result indicated that the single amino acid variation from D752 to N752 in DNA polymerase did not impair the in vivo growth of EHV-1 in mice, which was consistent with previous findings (Goodman et al., 2007). In contrast, in vivo replication of the gC-LI negative mutant 1_D752N AgC was significantly less effective (p < 0.001), with a mean viral titer of only 0.45 PFU/mg in lung tissues. On day 4 pi, no virus was recovered from infected LI 1_D752N AGC mice. With regard to histological changes at day 2 pi, the lungs of mice infected with either wild type or RacLl 1 or the mutant IL polymerase showed a D752N mild suppurative pneumonia accompanied by neutrophil infiltration, increased perivascular edema, and increased of inflammatory cells (lymphocytes predominantly) in the lymphatic vessels and perivascular area, whereas none were detected in lungs infected with LI 1_D752N AgC and PBS (Fig.10). As a result of the inflammatory response, continued body weight loss of mice infected with RacL1, LI 1_D752N or the rescuant virus LI 1_D752N AgC rev was observed until day 3 pi Body weight of mice infected with LI 1 D752N AgC, however, it did not show any apparent reduction. On day 28 pi, all mice were challenged intranasally with RacLl 1. Two days after challenge (p. One), RacLl 1 replicated to a titer of 2100 PFU/mg in the lungs of sham-inoculated mice, but only 18, 8 PFU/mg in LI 1_D752N AgC group (Fig.9). Virus titers in the lungs of mice inoculated with RacLl 1, LI or LI 1_D752N 1_D752N AGC rev were even lower (0.5, 0.9 and 1.3 PFU/mg, Fig.9) on day 2 p. c, which was, however, at the expense of high growth efficiency and in vivo pathogenicity. On day 4, p. c, re-isolation of the virus from the LI 1 D752N AgC group was not successful. Based on these results it is concluded that the GC-negative, nonneurological mutant LI 1_D752N AgC is severely attenuated and apathogenic to mice, but may confer protective immunity.

Example 5 - Vaccination of Horses

[0120] Horses (foals 6 to 8 months old) were grouped into three groups A, B and C. Group A was treated with live attenuated EHV-1 virus having the gC gene deleted (L11_D752N gC). Group B was treated with live attenuated EHV-1 virus (RacLl 1). Group C was the control group. Vaccination and sampling were performed according to the schedule presented in Table 2.

[0121] Both vaccines were well tolerated by the foals. Vaccinia virus strains could not be detected in any sample by virus isolation. Control horses did not seroconvert until the time of challenge, as evidenced by the absence of SN and CF antibody titers to EHV-1. Horses in groups A and B mounted an SN response to the first vaccination, which was boosted after the second vaccination. CF antibody titers in both groups of horses remained low/undetectable after the first vaccination and increased after the second vaccination, but did not reach high levels (Figure 11).

[0122] Clinical signs after challenge in the control horses included a tri-phasic fever response and respiratory illness characterized by moderate to severe nasal discharge. In contrast, hyperthermia was only found sporadically in both vaccinated groups of horses and nasal discharge was reduced in intensity and duration. A notable effect of the vaccine was the complete absence of viremia after challenge in 5/8 horses of both groups 1 and 2 (Figure 12). Furthermore, the duration of viremia was reduced in vaccinates compared to control horses. Figure 13 shows that group A had significantly reduced viral shedding in nasal swabs, whereas nasal shedding in group B was more variable but reduced compared to control horses. Example 6 - Sequencing a gC RacLl gene

[0123] The RacL1 gC gene has been sequenced and is represented by SEQ ID NO: 34 and SEQ ID NO: 35 for the DNA and protein sequence, respectively.

[0124] It is evident that the precise details of the methods described may be varied or modified without departing from the spirit of the disclosure described. We claim all such modifications and variations as fall within the scope and spirit of the claims below.

[00125] All documents cited or referenced ("referred to herein as cited documents"), and all documents cited or referenced herein cited documents, together with any manufacturer's instructions, descriptions, product specifications and product package inserts for all products herein or in any document mentioned herein incorporated by reference are incorporated herein by reference, and may be employed in the practice of the invention.

Claims (13)

1. Composition or vaccine characterized in that it comprises a recombinant Equine Herpesvirus-1 (EHV-1), in which the EHV-1 comprises a mutant gene, which encodes a non-functional glycoprotein C (gC), in which the nucleotides at positions 94 to 1315 inclusive of the gC gene of SEQ ID NO: 7, 9 or 11 are deleted. 2. Composition or vaccine, according to claim 1, characterized in that the recombinant EHV-1 comprises a DNA polymerase (Pol) that comprises an asparagine (N) at amino acid position 752. 3. Composition or vaccine, according to claims 1 or 2, characterized in that the EHV-1 is derived from an EHV-1 strain selected from the group consisting of RacH strain, RacL strain, Ab4 strain , strain V592, strain Kentucky D, strain 438/77, strain AB69, strain EHV-1 NY03 and a combination thereof. 4. Composition or vaccine according to any one of claims 1 to 3, characterized in that the EHV-1 is derived from the EHV-1 RacL strain, and in which the RacL DNA polymerase (Pol) comprises a asparagine (N) at amino acid position 752. 5. Composition or vaccine, according to any one of claims 1 to 4, characterized in that it further comprises a pharmaceutically or veterinarily acceptable vehicle, diluent, adjuvant or excipient. 6. Recombinant EHV-1 characterized in that it comprises a mutated gC gene, wherein the nucleotides at positions 94 to 1315 inclusive of the gC gene of SEQ ID NO: 7, 9 or 11 are deleted. 7. Recombinant EHV-1, according to claim 6, characterized in that the recombinant EHV-1 comprises a DNA polymerase (Pol) of the gene encoding a Pol comprising an asparagine (N) at amino acid position 752. 8. Recombinant EHV-1, according to claims 6 to 7, characterized in that the EHV-1 is derived from an EHV-1 strain selected from the group consisting of the RacH strain, the RacL strain, Ab4 strain, V592 strain, Kentucky D strain, 438/77 strain, AB69 strain, EHV-1 NY03 strain, and a combination thereof. 9. Recombinant EHV-1, according to claim 148, characterized in that EHV-1 is derived from the RacL strain of EHV-1. 10. Use of a composition or recombinant EHV-1 as defined by any one of claims 1 to 9 characterized in that it is for the preparation of a medicament for vaccination of an animal. 11. Use, according to claim 10, characterized in that it comprises a prime-boost regime. 12. Use of a composition or recombinant EHV-1 as defined by any one of claims 1 to 9 and a pharmaceutically or veterinary acceptable vehicle, carrier, adjuvant or excipient characterized in that it is for the preparation of a medicament to induce a protective response in an equine animal against Herpesvirus. 13. Use according to any one of claims 10 to 12, characterized in that the animal is an equine.

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